β1,3-Galactosyltransferase genes, derived from mammals [Japanese Published Unexamined Patent Application No. 181759/94; J. Biol. Chem., 272, 24794 (1997); J. Biol. Chem., 273, 58 (1998); Eur. J. Biochem., 263, 571 (1999); J. Neurosci. Res., 58, 318 (1999)] and derived from a bird [Acta Biochemica Polonica, 45, 451 (1998)] have been obtained. However, there is no example in which β1,3-galactosyltransferase gene derived from an animal was expressed as an active protein in a microorganism such as Escherichia coli. Although there is an example in which the gene derived from a bird was expressed in Escherichia coli [Acta Biochemica Polonica, 45, 451 (1998)], its activity was very weak.
On the other hand, in the case of microorganisms, a β1,3-galactosyltransferase gene has been obtained from Campylobacter jejuni and expressed in Escherichia coli as an active protein [J. Biol. Chem., 275, 3896 (2000); Mol. Microbiol., 37, 501 (2000)]. However, as the substrate of the protein, the references describe only GalNAcβ1,4[NeuAcα2,3]Galβ1,4Glc-FCHASE in which its non-reducing terminal has N-acetylgalactosamine and its reducing terminal is labeled with a fluorescent material [FCHASE (6-(5-fluoresceincarboxamido)hexanoic acid succimidyl ester)], and the references do not describe a complex carbohydrate having a modification on its reducing terminal or a complex carbohydrate having N-acetylglucosamine on its non-reducing terminal.
Regarding Helicobacter pylori 26695, the full nucleotide sequence of its genomic DNA has been determined [Nature, 388, 539 (1997)], but a gene product which functions as a β1,3-galactosyltransferase has not been known. Furthermore, since a Galβ1,3GlcNAc structure in Helicobacter pylori disappeared by disruption of a gene which corresponds to HP0619 gene of Helicobacter pylori 26695, BP0619 gene is expected to be a β1,3-galactosyltransferase gene [Infect. Immun., 68, 5928 (2000)], but there have been neither examples in which the gene was expressed in a microorganism such as Escherichia coli nor descriptions regarding its detailed enzyme activity.
Among galactose-containing complex carbohydrates, complex carbohydrates having a Galβ1,3-R structure in the skeleton are particularly important as synthesis intermediates of Lewisa or Lewisb which is a blood group complex carbohydrate [J. Pediatr. Gastroenterol. Nutr., 30, 181 (2000)], and is contained in human milk in a large amount and considered to have a preventive effect against bacterial infection [Anal. Biochem., 2239, 218 (1994); J. Chromatogr., 685, 211 (1996); Lancet, 347, 1017 (1996)].
For the production of the galactose-containing complex carbohydrates having a Galβ1,3-R structure, an extraction method from human milk [Carbohydr. Res., 178, 79 (1988)], a chemical synthesis method [Carbohydr. Res., 316, 121 (1999)] and the method using an enzyme [Glycoconjugate J., 16, 189 (1999)] have been reported, however, each of the methods has problems from the viewpoints of cost and productivity, and thus an industrial production method has not been established yet.